The field of the invention is generally related to methods of treating autoimmune diseases, including both antibody-mediated and cell-mediated disorders.
Autoimmune diseases are caused by the failure of the immune system to distinguish self from non-self. In these diseases, the immune system reacts against self tissues and this response ultimately causes inflammation and tissue injury. Autoimmune diseases can be classified into two basic categories: antibody-mediated diseases such as systemic lupus erythematosus (SLE), pemphigus vulgaris, myasthenia gravis, hemolytic anemia, thrombocytopenia purpura, Grave""s disease, Sjogren""s disease and dermatomyositis; and cell-mediated diseases such as Hashimoto""s disease, polymyositis, disease inflammatory bowel disease, multiple sclerosis, diabetes mellitus, rheumatoid arthritis, and scleroderma.
In many autoimmune diseases, tissue injury is caused by the production of antibodies to native tissue. These antibodies are called autoantibodies, in that they are produced by a mammal and have binding sites to the mammal""s own tissue. Some of these disorders have characteristic waxing and waning of the amount of circulating autoantibodies causing varying symptoms over time.
Of the different types of antibody-mediated autoimmune disorders, SLE is a disorder that has been well studied and documented. SLE is a disorder of generalized autoimmunity characterized by B cell hyperactivity with numerous autoantibodies against nuclear, cytoplasmic and cell surface antigens. This autoimmune disease has a multifactorial pathogenesis with genetic and environmental precipitating factors (reviewed in Hahn, B. H., Dubois"" Lupus Erythematosus, 5th Ed. (1997), pp. 69-76 (D. J. Wallace et al. eds., Williams and Wilkins, Baltimore)). Among the numerous lymphocyte defects described in SLE is a failure of regulatory T cells to inhibit B cell function (Horwitz, D. A., Dubois"" Lupus Erythematosus, 5th Ed. (1997), pp. 155-194 (D. J. Wallace et aL eds., Williams and Wilkins, Baltimore)). Sustained production of polyclonal IgG and autoantibodies in vitro requires T cell help (Shivakumar, S. et al. (1989), J Immunol 143:103-112).
Regulatory T cells can down-regulate antibody synthesis by lytic or cytokine-mediated mechanisms. The latter involve transforming growth factor-beta (TGF-xcex2) and other inhibitory cytokines (Wahl, S. M. (1994), J Exp Med 180:1587-190). Circulating B lymphocytes spontaneously secreting antibodies are increased in patients with active SLE (Klinman, D. M. et al. (1991), Arthritis Rheum 34:1404-1410).
Clinical manifestations of SLE include a rash (especially on the face in a xe2x80x9cbutterflyxe2x80x9d distribution), glomerulonephritis, pleurisy, pericarditis and central nervous system involvement. Most patients are women, and are relatively young (average age at diagnosis is 29).
The treatment of SLE depends on the clinical manifestations. Some patients with mild clinical symptoms respond to simple measures such as nonsteroidal anti-inflammatory agents. However, more severe symptoms usually require steroids with potent anti-inflammatory and immunosuppressive action such as prednisone. Other strong immunosuppressive drugs which can be used are azathioprine and cyclophosphamide. The steroids and other immunosuppressive drugs have side effects due to the global reduction of the mammal""s immune system. There is presently no ideal treatment for SLE and the disease cannot be cured.
Currently, considerable attention has been focused on the identity of genes which enhance the susceptibility or resistance to SLE, the identification of antigenic determinants that trigger the disease, the molecular mechanisms of T cell activation which results in survival or apoptosis, cytokines which determine T cell function, and the properties of the autoantibody-forming B cells. Many examples of T cell dysregulation in SLE have been described (reviewed in Horwitz, D. A. et al., Dubois"" Lupus Erythematosus, 5th Ed. (1997), pp. 83-96 (D. J. Wallace et al. eds., Williams and Wilkins, Baltimore). Although it is well recognized that the primary role of certain lymphocytes is to down-regulate immune responses, progress in elucidating the identity and mechanisms required for generation of these cells has been slow.
lnterleukin-2 (IL-2) has previously been considered to have an important role in the generation of antigen non-specific T suppressor cells. Anti-IL-2 antibodies given to mice coincident with the induction of graft-versus-host-disease resulted in several features of SLE (Via, C. S. et al. (1993), Intemational Immunol. 5:565-572). Whether IL-2 directly or indirectly is important in the generation of suppression has been controversial (Fast, L. D. (1992), J. Immunol.149:1510-1515; Hirohata, S. et al. (1989), J. Immunol. 142:3104-3112; Baylor, C. E. (1992), Advances Exp. Med. Biol. 319:125-135). Recently, IL-2 has been shown to induce CD8+ cells to suppress HIV replication in CD4+ T cells by a non-lytic mechanism. This effect is cytokine mediated, but the specific cytokine has not been identified (Kinter, A. L. et al. Proc. Natl. Acad. Sci USA 92:10985-10989; Barker, T. D. et aL (1996), J. Immunol. 156:4478-4483). T cell production of IL-2 is decreased in SLE (Horwitz, D. A. et al. (1997), Dubois"" Lupus Erythematosus, 5th Ed. (1997), pp. 83-96, D. J. Wallace et al. eds., Williams and Wilkins, Baltimore).
CD8+ T cells from subjects with SLE sustain rather than suppress polyclonal IgG production (Linker-Israeli, M. et al. (1990), Arthritis Rheum. 33:1216-1225). CD8+ T cells from healthy donors can be stimulated to enhance antibody production (Takahashi, T. et al. (1991), Clin. Immunol. Immunopath. 58:352-365). However, neither IL-2 nor CD4+ T cells, by themselves, were found to induce CD8+ T cells to develop strong suppressive activity. When NK cells were included in the cultures, strong suppressive activity appeared (Gray, J. D. et al. (1994) J. Exp. Med. 180:1937-1942). It is believed that the contribution of NK cells in the culture was to produce transforming growth factor beta (TGF-xcex2) in its active form. It was then discovered that non-immunosuppressive (2-10 pg/ml) concentrations of this cytokine served as a co-factor for the generation of strong suppressive effects on IgG and IgM production (Gray, J. D. et al. (1994) J. Exp. Med. 180:1937-1942). In addition, it is believed that NK cells are the principal source of TGF-xcex2 in unstimulated lymphocytes (Gray, J. D. et al. (1998), J. Immunol. 160:2248-2254).
TGF-xcex2s are a multifunctional family of cytokines important in tissue repair, inflammation and immunoregulation (Massague, J. (1980), Ann. Rev. Cell Biol 6:597). TGF-xcex2 is unlike most other cytokines in that the protein released is biologically inactive and unable to bind to specific receptors (Sporn, M. B. et al. (1987) J. Cell Biol. 105:1039-1045). The latent complex is cleaved extracelluarly to release active cytokine as discussed below. The response to TGF-xcex2 requires the interaction of two surface receptors (TGF-xcex2-R1) and TGF-xcex2-R2) which are ubiquitously found on mononuclear cells (Massague, J. (1992), Cell 69:1067-1070 ). Thus, the conversion of latent to active TGF-xcex2 is the critical step which determines the biological effects of this cytokine.
It was found that SLE patients have decreased production of TGF-xcex21 by NK cells. Defects in constitutive TGF-xcex2 produced by NK cells, as well induced TGF-xcex2 were documented in a study of 38 SLE patients (Ohtsuka, K. et al. (1998), J. Immunol. 160:2539-2545). Neither addition of recombinant IL-2 or TNF-alpha, or antagonism of IL-10 normalized the TGF-xcex2 defect in SLE. Decreased production of TGF-xcex2 in SLE did not correlate with activity of disease and, therefore, may be a primary defect.
Systemic administration of TGF-xcex2, IL-2, or a combination of both can lead to serious side effects. These cytokines have numerous effects on different body tissues and are not very safe to deliver to a patient systemically. It is, therefore, an object of the invention to provide methods and kits for treating mammalian cells that are responsible for controlling the regulation of autoantibodies to increase the population of cells that down regulate auto-antibody production.
In accordance with the objects outlined herein, the present invention provides methods for inhibiting immune responses in a sample of ex vivo peripheral blood mononuclear cells (PBMCs) comprising adding an regulatory composition to the cell population.
In an additional aspect, the present invention provides methods for treating an autoimmune disorder in a patient. The methods comprise removing peripheral blood mononuclear cells (PBMC) from the patient and treating the cells with an regulatory composition for a time sufficient to suppress inflamation and tissue injury. In particular, the methods of the present invention suppress antibody production or induce cells to down regulate antibody production and enhance cell mediated immune responses in patients with antibody mediated autoimmune diseases. The treated cells are then reintroduced to the patient, with a resulting amelioration of the autoimmune symptoms. The regulatory composition preferably comprises TGF-xcex2 and agents which enable T cells to respond to TGF-xcex2.
In an additional aspect, the present invention provides methods for treating cell-mediated autoimmune diseases. The methods comprise removing peripheral blood mononuclear cells (PBMC) from the patient and treating the cells with an regulatory composition for a time sufficient to suppress tissue injury by immune cells. The treated cells are then reintroduced to the patient, with a resulting amelioration of the autoimmune symptoms. The regulatory composition preferably comprises TGF-xcex2 and agents which enable T cells to respond to TGF-xcex2.
In an additional aspect, the invention provides kits for the treatment of an autoimmune disorder in a patient. The kits comprise a cell treatment container adapted to receive cells from a patient with an antibody-mediated autoimmune disorder or a cell-mediated disorder and at least one dose of an regulatory composition.